BENZYL ACETATE 1. EXPLANATION This substance was evaluated for an acceptable daily intake for man (ADI) by the joint FAO/WHO Expert Committee on Food Additives at the eleventh, twenty-seventh, twenty-ninth and thirty-first meetings (Annex 1, references 14, 62, 70 and 77). A toxicological monograph was issued in 1968 (Annex 1, reference 15). The ADI of 0-5 mg/kg bw/day which was established at the eleventh Meeting was made temporary at the twenty-seventh meeting. Since the previous evaluation, additional data have become available and are summarized and discussed in the following monograph. The previously published monograph has been expanded and is reproduced in its entirety below. 2. BIOLOGICAL DATA 2.1 Biochemical aspects 2.1.1 Absorption, distribution and excretion This compound is absorbed from the gastrointestinal tract, through the lungs and through the intact skin. It is hydrolyzed in man to benzyl alcohol and acetate: the benzyl radical is oxidized to benzoic acid and excreted as hippuric acid (Snapper et al., 1925). Benzyl acetate was readily hydrolyzed in vitro with a pancreatin preparation (Grundschoser, 1977). Benzylmercapturic acid and hippuric acid were isolated from the urine of rats that had been injected subcutaneously with benzyl acetate (Clapp & Young, 1970). Mice and rats were dosed either intravenously or orally with 14C labelled benzyl acetate. The intravenous dose for mice was equivalent to 10 mg/kg bw and for rats 5 mg/kg bw. For oral dosing, the benzyl acetate was dissolved in corn oil and administered at dose levels equivalent to 10, 100 or 1000 mg/kg bw for mice and 5, 50 and 500 mg/kg bw for rats. Elimination of benzyl acetate as CO2 or volatiles was minimal. The elimination of the 14C label occurred mainly in the urine with less than 1% being detected in the feces. More than 90% of the 14C in the urine was present as hippuric acid, with minor amounts present as benzyl alcohol and benzylmercapturic acid (up to 4%). No unchanged benzyl acetate was present in urine. The elimination was complete within 24 hours. Neither route of administration or dose had any significant effect on the pattern of elimination. Benzoyl glucuronide levels were not measured. Repeated dosing of rats with 500 mg/kg bw/day benzyl acetate for 14 days, followed by a single dose of 14C benzyl acetate did not change the clearance pattern. Analysis of tissues of animals, sacrificed 24 hours after administration of 14C either i.v. or orally, failed to detect the presence of 14C activity (Matthews & Burka, 1984). Male rats received [methylene-14C]-benzyl acetate by gavage either in corn oil or in propylene glycol at doses of 5, 250 or 500 mg/kg bw. Radioactivity was measured in feces, urine, plasma and tissues. Metabolites in urine and plasma were identified by HPLC and TLC. Absorption of benzyl acetate was more rapid at the lower doses and in the absence of a vehicle. The bulk of the administered dose (70-89%) was excreted in the urine over the initial 24-hr period with very little in the feces (approximately 4%) after 72 hours. The elimination of benzyl acetate and metabolites was essentially complete by 3 days, as indicated by negligible residues in tissue, regardless of whether benzyl acetate was given neat or in corn oil. Since benzyl acetate was not found in the plasma or urine at any time, the authors concluded that it was readily hydrolyzed. Small amounts of benzyl alcohol, the initial product of ester cleavage, was detected only in plasma samples. At the higher doses, benzoic acid was the major metabolite in plasma, while at the 5 mg/kg bw dose, hippuric acid (the glycine conjugate of benzoic acid) predominated. Hippuric acid was the major metabolite in urine, and the proportion of the original dose it represented was not significantly affected by dose size. The proportion of the dose present as benzoyl glucuronide increased with dosage, leading the authors to conclude that this demonstrated a limited capacity of the glycine conjugation mechanism (Chidgey & Caldwell, 1986). 2.2 Toxicological studies 2.2.1 Acute toxicity Species Sex Route LD50 Reference (mg/kg bw) Rat ? oral 2490-3690 Jenner et al., 1967 von Oettingen, 1960 Rabbit ? oral 2640 von Oettingen, 1960 2.2.2 Short-term studies 2.2.2.1 Mice Groups of 5 mice (B6C3F1) of each sex were administered single doses of benzyl acetate equivalent to 250, 500, 1000, 2000 or 4000 mg/kg bw in corn oil by gavage. The animals were observed for 15 days. All mice receiving 4000 mg/kg bw and females receiving 2000 mg/kg bw became inactive immediately after dosing. All mice in the highest dose group, and 1/5 males and 2/5 females dosed at 2000 mg/kg bw died by day 2 of the study. No other compound-related effects were reported (NTP, 1986). Groups of 5 mice (B63F/N) of each sex were dosed with 0, 125, 250, 500, 1000 or 2000 mg/kg bw benzyl acetate in corn oil by gavage daily for 14 days. On day 16 all surviving animals were killed and autopsied. All male mice at the highest dose level had died by day 3 of the study. Weight changes were not dose-related. At autopsy the only effect reported was a roughening of the mucosa of the stomach in the cardiac region in 2/5 males and all females in the highest dose group, and 1/5 females in the 1000 mg/kg bw/day group (NTP, 1986). Groups of 10 mice (B6C3F1) of each sex were administered 0, 125, 250, 500 or 12000 mg/kg bw/day benzyl acetate in corn oil by gavage, 5 times a week for 13 weeks. A total of 7/10 of the females in the highest dose group died. Compound-related clinical effects were observed in the highest dose group, included trembling, inactivity, labored breathing and depressed body temperature. At autopsy, no gross or microscopic effects were noted (NTP, 1986). 2.2.2.2 Rats Groups of 15 males and 15 females were fed a mixture of aromatic esters, including 15.8 mg/kg bw/day of benzyl acetate for 12 weeks. No adverse effects were noted (Oser, 1967). Groups of 5 rats (F344/N) approximately 6 weeks old, were administered 0, 250, 500, 1000, 2000 or 4000 mg/kg bw/day benzyl acetate by gavage in corn oil, daily for 14 days. On day 16, surviving animals were killed and autopsied. None of the rats dosed at 4000 mg/kg bw/day survived beyond 2 days, at the 2000 mg/kg bw/day level all rats had died within 5 days. No other deaths were reported. There was a depression of mean body weight gain relative to controls of more than 10% in the 500 mg/kg bw/day male group, and in the 1000 mg/kg bw/day group in both males and females. At autopsy the only effect reported was that the cecum was redder than normal in 3/5 of the animals in the 4000 mg/kg bw/day group (NTP, 1986). Groups of 10 rats (F344/N) of each sex were administered 0, 62.5, 125, 250, 500 or 1000 mg/kg bw/day benzyl acetate in corn oil, 5 days per week for 13 weeks. Male and female rats in the 100 mg/kg bw/day group and females in the 500 mg/kg bw/day group showed clinical symptoms including trembling, ataxia and sluggishness. A total of 2/10 males and 1/10 females in the highest dose group had died by day 86. Only male rats in the 1000 mg/kg bw/day group showed a depression in mean body weight relative to controls (21%). At autopsy a thickened stomach wall was observed in 2/9 males and 4/10 females in the high dose group (NTP, 1986). 2.2.3 Long-term/carcinogenicity studies Lifetime studies in mice and rats with benzyl alcohol are included in this section since the initial step in metabolism of benzyl acetate is hydrolysis of the ester to benzyl alcohol (Abdo et al., 1985; Chidgey et al., 1986; NTP, 1986). 2.2.3.1 Mice Groups of 50 (B6C3F1) mice of each sex were administered benzyl acetate in corn oil by gavage at doses equivalent to 500 or 1000 mg/kg bw/day, 5 days/week for 103 weeks. Vehicle control groups of 50 animals/sex were administered corn oil by gavage. Complete gross necropsies and histopathological examinations were performed on animals found dead and on those sacrificed at the end of the study. Mean body weight gains of dosed and control male mice were comparable throughout the study. In females, treated mice showed slightly higher mean body weights after week 20 than those of the controls. Survival of female mice was markedly reduced in the control (30%) and low dose groups (36%) in comparison with the high dose group (60%). The high mortality rate in female mice was associated with infection, resulting in suppurative inflammation or abscesses of the ovaries, uterus, mesentery, peritoneum or multiple organs (26/35 control, 14/32 low dose and 8/20 high dose). In males, there were no significant differences between the survival rate of controls and either treatment group, although a greater number of control males died before week 45 than in the treated groups. Hepatocellular adenomas occurred in male mice in 0/50 control, 5/49 low dose and 13/50 high dose animals, and in female mice in 0/50 control, 0/50 low dose, and 6/50 high dose animals. (The cumulative historical incidences of hepatocellular adenomas in corn oil gavage controls from 6 studies from the contract laboratory conducted prior to 3 August 1984, were 36/298 for male and 11/300 for female B6C3F1 mice). Hepatocellular carcinomas were observed in male mice and in the high dose female group (Males: control 10/50, low dose 14/49 and high dose 12/50, and females: 1/50 control, 0/50 low dose, and 4/50 high dose). Squamous cell papillomas or carcinomas of the forestomach occurred in male mice and in the high dose female group. The incidence in males was 4/49 control, 4/48 low dose, and 11/49 high dose. For female mice the incidence was 0/50 control, 0/50 low dose and 4/48 high dose. The historical incidences in corn oil gavage controls from 6 studies from the contract laboratory were 2/296 for males and 2/297 for females. Forestomach hyperplasia was also reported in dosed mice (males: control 1/49, low dose 7/48 and high dose 22/49, and for females: control 1/50, low dose 6/50, high dose 17/48) (NTP, 1986). Benzyl alcohol was administered by corn oil gavage to groups of 50 mice (B6C3F1) of either sex, at dosages of 0, 100 or 200 mg/kg bw/day, 5 days a week for 103 weeks. The neurotoxic effects of benzyl alcohol (lethargy and staggering) in short-term studies formed the basis for selection of the doses. Complete gross necropsies and histopathological examinations were conducted on animals found dead and on those sacrificed at the end of the study. Mean body weights for dosed and control mice were comparable throughout the study. The survival of control females was significantly lower than those in the high dose group after week 44, but no other differences in survival were noted between dosed groups and controls (male: control 68%, low dose 66%, high dose 70%; female: control 50%, low dose 62%, high dose 72%). No significant treatment-related effects were noted. There was no association of treatment with the reported incidences of hepatocellular and forestomach neoplasias after administration of benzyl acetate (NTP, 1987). 2.2.3.2 Rats Groups of 50 rats (F344/N) of each sex were administered benzyl acetate in corn oil by gavage at doses equivalent to 150 or 500 mg/kg bw/day, 5 days/week for 103 weeks. Vehicle control groups of 50 animals per sex were administered corn oil by gavage. Complete gross necropsies and histopathological examinations were performed on animals found dead and on those sacrificed at the end of the study. Mean body weight gains were comparable between treated and control groups throughout most of the study. No significant differences were found in the survival of male and female rats administered benzyl acetate as compared with controls. In male rats, 76% of the controls, 92% of the low-dose group and 80% of the high- dose group survived to 104-106 weeks. Survival rates for females at 104-106 weeks were: 80% of the controls, 72% of the low-dose group and 72% of the high-dose group. The incidence of all malignant epithelial tumours (cystadenocarcinoma, adenocarcinoma and carcinoma) in the preputial gland of male rats was elevated in the high dose group (control 1/50, low dose 1/50, high dose 6/50), but this was without statistical significance. The incidence of acinar cell adenomas in the pancreas of male rats was 22/50 in controls, 27/50 at the low dose, and 37/49 at the high dose. The incidence at the high dose was significantly greater than in the vehicle controls (p = 0.001). Acinar cell hyperplasia was also observed in male rats (37/50 control, 34/50 low dose, and 36/49 high dose). No acinar cell hyperplasia or adenoma of the pancreas was observed in any of the female animals in the three experimental groups. There was an increased incidence of retinopathy (not specified) and cataracts in the high dose males and low dose females. However, this effect was attributed by the authors to the proximity of these rats to fluorescent light (NTP, 1986). Benzyl alcohol was administered by corn oil gavage to groups of 50 F344/N rats of each sex at dosages of 0, 200 or 400 mg/kg bw/day, 5 days a week for 103 weeks. Complete gross necropsies and histopathological examinations were conducted on animals found dead and on those sacrificed at the end of the study. Mean body weights of dosed and vehicle control male and female rats were comparable throughout the study. No compound-related clinical signs were observed, although at month 3 a sialodacryoadenitis virus infection was widespread among the study animals. The survival of animals in both dosed female groups was significantly lower than that of vehicle controls (70% - control; 34% - low dose; 34% - high dose), which was the result of a much higher incidence of accidental gavage-related deaths in these groups. Survival among male rats was comparable in all groups (control - 56%; low dose - 54%; high dose - 48%). Cataracts and retinal atrophy were observed at increased incidences in the high dose rats in this study. This was again attributed by the authors to the proximity of this group of animals to fluorescent light for most of the study. Hyperplasia of the forestomach epithelium was noted at an increased incidence in male rats (control 0/48, low dose 0/19, high dose 4/50). Hemorrhage and foreign material in the respiratory tract were noted in dosed rats which died prior to the terminal kill. The authors suggested this could have been the result either of direct deposition of material into the lung due to gavage "accidents" or of the anesthetic properties of benzyl alcohol resulting in reflux of gavage material, and aspiration into the lungs. No pancreatic acinar cell adenomas were reported in any of the test or control groups in contrast to the results from the corn oil gavage study in rats with benzyl acetate. No other effects of treatment were noted (NTP, 1987). 2.2.5 Special studies on genotoxicity Results of genotoxicity assays on benzyl acetate Test System Test Object Concentration Results Reference of benzyl acetate Ames test (1) S.typhimurium 0-10,000 µg/ Negative NTP,1986; TA100,TA1535, plate Mortelmans TA1537, TA98 et al., 1986 In vitro Chinese 50-5,000 µg/ml Negative NTP, 1986 mammalian cytogenicity hamster (SCE, chromosomal ovary cells aberrations) (2) In vitro Mouse lymphoma 0.25-1.25 µg/ml Positive NTP, 1986 mammalian gene cells (3) mutation (2) (L5178Y/Tk±) In vitro Bacillus 21 µg/disk Negative Oda et al., bacterial subtilis 1978 gene mutation Unscheduled DNA Rat hepatocytes Negative Mirsalis et al., synthesis 1983 in vivo and in vitro) (1) in the presence or absence of induced rat or hamster liver S-9 fractions (2) in the presence or absence of induced rat liver S-9 fraction (3) positive in the presence of metabolic activation, negative in the absence of metabolic activation. 3. COMMENTS At the thirty-first meeting the Committee extended the temporary ADI of 0-5 mg per kg bw pending the evaluation of lifetime gavage studies with benzyl alcohol, a normal metabolite of benzyl acetate. These studies did not reveal the increased incidence of either hepatocellular or forestomach tumours in mice or pancreatic tumours in rats which had previously been observed in studies with benzyl acetate. There are difficulties in interpreting the carcinogenicity studies with benzyl acetate that were conducted by gavage. Since it is known that new long-term studies are underway with benzyl acetate incorporated into the diet of rats and mice, the present Committee decided to extend the temporary ADI of 0-5 mg/kg bw until 1993 pending the evaluation of the results of the new studies. In view of one positive report of mutagenic activity in vitro, it would be desirable to ascertain whether results of an in vivo study demonstrating the lack of the induction of unscheduled DNA synthesis can be confirmed by an in vivo test for chromosome damage in bone marrow. 4. EVALUATION Level causing no toxicological effect Rat: 15.8 mg/kg bw/day. Estimate of temporary acceptable daily intake 0-5 mg/kg bw. Further work or information Required (by 1993): Oncogenicity studies in rats and mice. Desired: In vivo study for chromosome damage in bone marrow. 5. REFERENCES ABDO, K.M. HUFF, J.E., HASEMAN, J.K., BOORMAN, G.A., EUSTIS, S.L., MATTHEWS, H.B., BURKA, L.T., PREJEAN, J.D. & THOMPSON, R.B. (1985). Benzyl acetate carcinogenicity, metabolism and disposition in Fischer 344 rats and B6C3F1 mice. Toxicology, 37, 159-170. CHIDGEY, M.A.J. & CALDWELL, J. (1986). Studies on benzyl acetate. I. Effect of dose size and vehicle on the plasma pharmacokinetics and metabolism of [methylene-14] benzyl acetate in the rat. Food Chem.Toxic., 2, 1257-1265. CHIDGEY, M.A.J., KENNEDY, J.F. & CALDWELL, J. (1986). Studies on benzyl acetate. II. Use of specific metabolic inhibitors to define the pathway leading to the formation of benzylmercapturic acid in the rat. Food Chem.Toxicol., 24, 1267-1271. CLAPP, J.J. & YOUNG, L. (1970). Formation of mercaptic acid in rats after administration of aralkyl esters. Biochem.J., 118, 765-771. GRUNDSCHOSER, F. (1977). Toxicological assessment of flavouring esters. Toxicology, 8, 387-390. JENNER, P., HAGAN, E., TAYLOR, J., COOK, E. & FITZHUGH, O.G. (1967). Food flavourings and compounds of related structure, I. Acute Oral Toxicity. Food Cosmet.Toxicol., 2, 327-343. MATTHEWS, H.B. & BURKA, L.T. (1984). Benzyl acetate metabolism and disposition in rats and mice, NTP (1984). NIH Publication No. 82-2506, USDHSS. MIRSALIS, J., TYSON, K., BECK, J., LOH, F., STEINMETZ, K., CONTRERAS, C., AUSTERE, L., MARTIN, S. & SPALDING, J. (1983). Induction of unscheduled DNA synthesis (UDS) in hepatocytes following in vitro and in vivo treatment (Abstract No. Ef-5). Environ.Mutagenesis, 5, 482. MORTELMANS, K., HAWORTH, S., LAWLOR, T., SPECK, W., TAINER, B. & ZEIGER, E. (1986). Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals. Environmental Mutagenesis, 8, 1-119. NTP (1986). NTP technical report on the toxicology and carcinogenesis studies of benzyl acetate (CAS NO. 140-11-4) in F344/N rats and B6C3F1 mice (gavage studies). NIH Publication No. 86-2506, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. NTP (1987). Board Draft. NTP technical report on the toxicology and carcinogenesis studies of benzyl alcohol (CAS NO. 100-51-6) in F344/N (gavage studies). NIH Publication No. 88-2599, U.S. Department of Health and Human Services, Public Health Service, National Institutes of Health, Research Triangle Park, NC. ODA, Y., HAMANO, Y., INQUE, K., YAMAMOTO, H., NIIHARA, T. & KUNITA, N. (1978). Mutagenicity of food flavours in bacteria. Osaka-furitsu Kosnu Eisei Kenkyu Hokoku snokunin eisein nem, 9, 177-181. OSER, B.L. (1967). Unpublished report. SNAPPER, I., GRUNBAUM, A. & STRUKOP, S. (1925). Biochem.Z., 155, 163. VON OETTINGEN, W.W. (1960). AMA Arch.Ind. Health, 21, 28.
See Also: Toxicological Abbreviations Benzyl acetate (ICSC) Benzyl acetate (FAO Nutrition Meetings Report Series 44a) Benzyl acetate (WHO Food Additives Series 32) Benzyl acetate (WHO Food Additives Series 37) BENZYL ACETATE (JECFA Evaluation) Benzyl Acetate (IARC Summary & Evaluation, Volume 40, 1986) Benzyl Acetate (IARC Summary & Evaluation, Volume 71, 1999)